1Department of Medicine (Gastroenterology), University of Alabama at Birmingham, Birmingham, Alabama, United Sates of America.

Abstract

Tissue macrophages are derived exclusively from blood monocytes, which as monocyte-derived macrophages support HIV-1 replication. However, among human tissue macrophages only intestinal macrophages are non-permissive to HIV-1, suggesting that the unique microenvironment in human intestinal mucosa renders lamina propria macrophages non-permissive to HIV-1. We investigated this hypothesis using blood monocytes and intestinal extracellular matrix (stroma)-conditioned media (S-CM) to model the exposure of newly recruited monocytes and resident macrophages to lamina propria stroma, where the cells take up residence in the intestinal mucosa. Exposure of monocytes to S-CM blocked up-regulation of CD4 and CCR5 expression during monocyte differentiation into macrophages and inhibited productive HIV-1 infection in differentiated macrophages. Importantly, exposure of monocyte-derived macrophages simultaneously to S-CM and HIV-1 also inhibited viral replication, and sorted CD4+ intestinal macrophages, a proportion of which expressed CCR5+, did not support HIV-1 replication, indicating that the non-permissiveness to HIV-1 was not due to reduced receptor expression alone. Consistent with this conclusion, S-CM also potently inhibited replication of HIV-1 pseudotyped with vesicular stomatitis virus glycoprotein, which provides CD4/CCR5-independent entry. Neutralization of TGF-β in S-CM and recombinant TGF-β studies showed that stromal TGF-β inhibited macrophage nuclear translocation of NF-κB and HIV-1 replication. Thus, the profound inability of intestinal macrophages to support productive HIV-1 infection is likely the consequence of microenvironmental down-regulation of macrophage HIV-1 receptor/coreceptor expression and NF-κB activation.

CD4+ intestinal macrophages and blood monocytes were purified from jejunal and blood MNLs from the same donor by MACS sorting, cultured for 4 days, inoculated with highly macrophage-tropic R5 viruses (MOI = 1) and monitored for p24 production at 4-day intervals for 20 days (n = 2 donors, p24 determinations for each donor in triplicate). Inset dot plots show staining with isotype control antibodies. The % in the histograms indicates percentage of CCR5+ cells among CD13+CD4+ cells.

(A) Monocyte-derived macrophages were generated by culturing monocytes for 2 days in media alone (control) or media plus increasing concentrations of stoma-conditioned media (S-CM) derived from normal jejunum from 2 different tissue donors and then analyzed for CD4 and CCR5 expression by flow cytometry. Data are from a representative experiment from 4 blood monocyte donors. (B) Monocyte-derived macrophages were generated by culturing monocytes for 4 days in media alone (control), after which S-CM (100 µg protein/mL) was added for an additional 2 days of culture, and the cells were analyzed for CD4 and CCR5 expression. Values are the % cells positive for the indicated receptor in a representative experiment from 3 blood monocyte donors.

(A) MACS-sorted monocytes were cultured for 4 days in M-CSF and the resultant monocyte-derived macrophages were cultured for an additional 2 days in the presence of S-CM at the indicated concentrations, then inoculated with R5 virus (NA353 B27; MOI = 1) for 2 hours and monitored for p24 release at 4-day intervals for 20 days (n = 4 donors, each in triplicate). (B) Monocyte-derived macrophages were cultured for 2 days with intestinal epithelial cell-conditioned media (EC-CM), mononuclear leukocyte (MNL)-CM or S-CM derived from the same normal jejunal tissue at the indicated concentrations, inoculated with R5 virus in triplicate as above, and analyzed for p24 release on day 12 (mean ± SD; n = 3). (C) Monocyte-derived macrophages prepared as in A were inoculated simultaneously with R5 virus (NA353 B27; MOI = 1) and S-CM, cultured for 2 hrs and then monitored for p24 release as in A (n = 4 donors, each in triplicate).

Monocytes were cultured for 4 days, inoculated with VSV-G or YU2 env pseudotyped GFP reporter virus and cultured for an additional 2 days, after which GFP expression was analyzed by fluorescence microscopy and flow cytometry in a representative experiment using monocyte-derived macrophages and S-CM from separate donors (n = 3).

Down-regulation of NF-κB by S-CM correlates with down-regulation in the cells' ability to support HIV-1 replication.

(A) Monocytes were cultured in media plus M-CSF and then inoculated in triplicate with R5 HIV-1 (NA353 B27; MOI = 1) plus S-CM at the indicated concentration for 2 hours. Cells were evaluated for NF-κB p65 translocation by confocal microscopy and NF-κB intensity by IPLab image analysis software after 2 hours and for viral replication by p24 ELISA on day 12 (n = 3 donors). Histograms are representative of a single experiment and show distribution of NF-κB (green line) in relation to the nucleus (blue line). The p24 value of each treatment was normalized to the media control group with the replication level of media control group defined as 100%. Data shown are the means of relative p24 levels from independent experiments with 3 donors. (B) Anti-TGF-β antibodies reverse the inactivation of NF-κB and S-CM-mediated down-regulation of HIV-1 replication. Experiments were performed as in A except the S-CM (250 µg protein/mL) was pre-incubated for 1 hour with anti-TGF-β antibodies at the indicated concentration (n = 3 donors). (C) Recombinant human TGF-β reduces NF-κB translocation and R5 virus replication. Experiments were performed as in A, except the S-CM was replaced with rhTGF-β at 10 or 50 pg/mL (n = 4 donors).